The Unit on Protein Biogenesis studies the mechanisms regulating the synthesis, translocation and maturation of secretory and membrane proteins at the mammalian endoplasmic reticulum (ER). A complex macromolecular assembly at the ER, termed the translocon, serves as a protein-conducting channel where substrates enter the secretory pathway. The translocon participates in diverse cellular activities that range from the import of secretory proteins, to topogenesis and assembly of complex multi-spanning membrane proteins, to the export of misfolded substrates from the ER to the cytosol for degradation. A largely unexplored aspect of ER function is the question of where within the ER these various events occur. Are all of these translocon-associated activities homogeneously distributed throughout the ER, or are they organized and regulated in the spatial and temporal dimensions to meet the changing needs of the cell? There is presently little or no insight into this question, largely because the current approaches to understanding protein translocation utilize biochemical systems in which spatial relationships are lost. In collaboration with the laboratory of Jennifer Lippincott-Schwartz, we are utilizing biophysical techniques such as fluorescence resonance energy transfer (FRET) to probe, in situ, the molecular organization of the components of the translocation machinery. In initial studies, analysis of FRET between subunits of the Sec61p complex, a principal component of the ER protein translocon, was used to directly monitor the assembly state of the translocon in cells. Our studies have revealed that while the translocon can be assembled from its components in response to ligands for protein translocation in biochemical systems, it does not disassemble and reassemble between successive rounds of transport in vivo. Instead, an actively engaged translocon is distinguished from a quiescent translocon by conformational changes that can be directly detected by differences in FRET. By correlating the formation of particular protein complexes with biochemical activities, we endeavor to directly visualize the functional segregation and organization of the ER, and to monitor potential changes in it during cellular metabolism, development, or disease pathogenesis.